Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD), and apoE4 carriers account for 65-80% of AD cases. ApoE4 increases the occurrence and lowers the age of onset of AD, and considerable evidence suggests that it has a fundamental role in the neurodegeneration and pathophysiology of AD. We showed that apoE4, because of its unique structural feature referred to as apoE4 domain interaction, is highly susceptible to proteolytic cleavage in neurons, generating neurotoxic fragments. The primary toxic fragment, apoE4(1-272), is found at much higher levels in the brain and cerebrospinal fluid of AD patients than non-demented individuals and in transgenic mice expressing human apoE4 in neurons. These transgenic mice have learning and memory impairments and neuropathology, including loss of synaptodendritic connections, which correlates with the onset of learning deficits. The toxicity induced by apoE4 and apoE4(1-272) is mediated by their inhibition of mitochondrial function, an initial event leading to neurodegeneration and cognitive impairment in AD. Thus, a number of studies have validated apoE4 as an excellent target for AD drugs. We discovered small molecules that bind apoE4 to modify the protein's structure to reduce domain interaction and proteolytic cleavage to prevent its toxicity. Our lead apoE4 structure corrector (apoE4SC), PY-101, protects neurons from apoE4-induced mitochondrial toxicity and reverses the impairment of neurite outgrowth in vitro. In vivo, PY-101 has good pharmacokinetics (PK) and brain bioavailability, blocks formation of toxic apoE4(1-272) in neuron-specific enolase (NSE)-apoE4 transgenic mouse brain, and protects against mitochondrial impairment in brain, an initial event in disease progression in AD. In this U01 grant, we propose to conduct the preclinical studies to evaluate the efficacy of PY-101 as a unique drug to treat AD. We will test PY-101 for efficacy in blocking cognitive impairment and neuropathology in the NSE-apoE4 and APP/apoE4 animal models of AD to validate PY-101 as a drug to treat AD. In addition, we will focus on optimizing the properties of apoE4SCs using our pharmacophore model based on the structure-activity relationship around PY-101. We will use this model to build a chemical optimization program around a novel chemical series to improve the potency of our prototypical apoE4SC while maintaining good PK and high brain penetration. Using the model, medicinal chemistry, and our high-throughput screening assays, we will optimize the efficacy and pharmaceutical properties of our apoE4SCs. To do this, small molecules will be screened against our primary GFP-apoE4-eDHFR FRET assay, which measures target engagement and the ability to disrupt apoE4 domain interaction to increase apoE4 stability. Optimized apoE4SCs will be subjected to stability, PK, and pharmacodynamic studies to select leads to test for efficacy in AD mouse models. The lead apoE4SC will be subjected to standard investigational new drug-enabling preclinical development with a goal of initiating clinical studies to test for safety and efficacy in treating D and proceeding to an IND.